Identification of S-Nitrosylation Motifs by Site-Specific Mapping of the S-Nitrosocysteine Proteome in Human Vascular Smooth Muscle Cells

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Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells

Todd M. Greco*, Roberto Hodara*, Ioannis Parastatidis*, Harry F. G. Heijnen†, Michelle K. Dennehy‡, Daniel C. Liebler‡, and Harry Ischiropoulos*§

*Stokes Research Institute and Departments of Pediatrics and Pharmacology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104; †Thrombosis and Haemostasis Laboratory, Department of Cell Biology, University Medical Center Utrecht, and Institute for Biomembranes, 3584 CH, Utrecht, The Netherlands; and ‡Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved March 22, 2006 (received for review January 27, 2006) S-nitrosylation, the selective modification of cysteine residues in critical roles for nitric oxide, the targets of S-nitrosylation in proteins to form S-nitrosocysteine, is a major emerging mechanism vascular smooth muscle cells are largely unknown. To that end, by which nitric oxide acts as a signaling molecule. Even though proteomic approaches are highly informative in providing a global nitric oxide is intimately involved in the regulation of vascular assessment of the modified proteins in cells and tissues. smooth muscle cell functions, the potential protein targets for Proteomic approaches based on the biotin-switch method have nitric oxide modification as well as structural features that underlie been used to identify potential targets of S-nitrosylation in various the specificity of protein S-nitrosocysteine formation in these cells model systems including murine brain tissue (15) and RAW 264.7 remain unknown. Therefore, we used a proteomic approach using cells (16), Mycobacterium tuberculosis (17), mouse mesangial cells selective peptide capturing and site-specific adduct mapping to (18), and human aortic endothelial cells (19, 20), yet the structural identify the targets of S-nitrosylation in human aortic smooth features that subserve the specificity of S-nitrosylation remain muscle cells upon exposure to S-nitrosocysteine and propylamine contentious. Recently, a peptide capture approach simultaneously propylamine NONOate. This strategy identified 20 unique S-ni- identified 68 unique S-nitrosocysteine residues belonging to 56 trosocysteine-containing peptides belonging to 18 proteins includ- proteins from S-nitrosoglutathione-treated rat cerebellar lysates ing cytoskeletal proteins, chaperones, proteins of the translational (21). Analysis of the identified peptides by a machine learning machinery, vesicular transport, and signaling. Sequence analysis of approach did not reveal linear sequence motifs under the experi- the S-nitrosocysteine-containing peptides revealed the presence of mental conditions used (21). However, subsequent inspection of the acid͞base motifs, as well as hydrophobic motifs surrounding the identified peptides indicated a prevalence for an acid͞base motif, identified cysteine residues. High-resolution immunogold electron suggesting that exploration of additional S-nitrosocysteine pro- microscopy supported the cellular localization of several of these teomes may further clarify the structural motifs that underlie the proteins. Interestingly, seven of the 18 proteins identified are specificity of S-nitrosylation. localized within the ER͞Golgi complex, suggesting a role for To this end, in intact human aortic smooth muscle cells S-nitrosylation in membrane trafficking and ER stress response in (HASMC) exposed to S-nitrosocysteine (CysNO) or propylamine vascular smooth muscle. propylamine NONOate (PAPANO), we identified potential targets of S-nitrosylation and evaluated S-nitrosylation motifs under con- nitric oxide ͉ proteomics ͉ S-nitrosothiols ditions that preserve the cellular localization of proteins as well as endogenous protein–protein interactions. Using a proteomic approach that selectively identified the modified S-nitrosocysteine -nitrosylation, the formal transfer of nitrosonium to a reduced residues, 18 proteins were identified. The localization of several of cysteine, is a reversible and selective posttranslational modifi- S these proteins was further supported by high-resolution immuno- cation that regulates protein activity, localization, and stability, and gold electron microscopy. Primary sequence analysis of the S- also functions as a general sensor for cellular redox balance (1–7). nitrosocysteine-containing peptides revealed the presence of acid͞ The formation of protein S-nitrosocysteine requires the removal of base motifs as well as the occurrence of cysteine residues within a single electron, i.e., the conversion of the nitrogen in nitric oxide hydrophobic pockets. from an oxidation state of 2 to 3. Several distinct pathways could satisfy the formation of protein S-nitrosocysteine adducts in bio- Results and Discussion logical systems, such as autooxidation of nitric oxide forming higher Formation of S-Nitrosocysteine Protein Adducts in Human Aortic oxides of nitrogen, radical recombination of thiyl radical with nitric Smooth Muscle Cells. The intracellular protein S-nitrosocysteine oxide, catalysis by metal centers, the direct reaction of nitric oxide content was evaluated by reductive chemistries coupled with chemi- with a reduced cysteine followed by electron abstraction, and luminescence detection (22). Naı¨ve HASMC in culture had levels transnitrosation reactions carried out by S-nitrosoglutathione, of protein S-nitrosocysteine below the lower limits of detection, and other small molecular mass S-nitrosothiols, and more recently, Western blot analysis failed to document expression of nitric oxide S-nitrosocysteine-containing proteins (8–10). synthases in these cells (not shown). Therefore, to generate endog- In vascular smooth muscle cells, nitric oxide derived from enous S-nitrosylated proteins, intact cells were exposed to either endothelium regulates important biological functions beyond re- laxation, such as phenotypic changes, proliferation, and commit- ment to undergo apoptosis (11, 12). Previous studies have shown Conflict of interest statement: No conflicts declared. that the molecular mechanisms underlying the functions of nitric This paper was submitted directly (Track II) to the PNAS office. oxide in vascular smooth muscle are mediated by both soluble Abbreviations: HASMC, human aortic smooth muscle cell; CysNO, S-nitrosocysteine; PA- guanylate cyclase-dependent and independent mechanisms (12– PANO, propylamine propylamine NONOate; HPDP–biotin, N-[6(Biotinamido)hexyl]-3Ј-(2Ј- 14). It has been suggested that selective S-nitrosylation of protein pyridyldithio) propionamide; MS͞MS, tandem MS. targets are responsible for the guanylate cyclase-independent reg- §To whom correspondence should be addressed. E-mail: [email protected]. ulation of vascular smooth muscle cell biology (13). Despite these © 2006 by The National Academy of Sciences of the USA

7420–7425 ͉ PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600729103 Downloaded by guest on October 1, 2021 Fig. 1. Evaluation of SEQUEST peptide assignments. (A) An MS͞MS spectrum (XCorr 3.6) assigned to an S- nitrosocysteine-containing peptide from 14-3-3 protein ␨ that met all selection criteria and was accepted. (B)An MS͞MS spectrum (Xcorr 4.1) assigned to a peptide from vimentin. Although this assignment passed the initial selection criteria, it was ultimately rejected because the top three most intense fragment peaks were not assigned (arrow). The evaluation of SEQUEST peptide assignments was assessed by multiple selection criteria as fol- lows: (i) Only peptide assignments that identified a biotin-HPDP derivitized cysteine (ϩ428) included in the y-orb-ion series were considered. (ii) Each experimental condition was performed in quadruplicate, with peptide assignments evaluated if they appeared in at least three of the four independent replicates. (iii) Peptide assignments that passed these two selection filters were then evaluated by output scores assigned by SEQUEST and were rejected if they did not meet specific threshold values as described in the Materials and Methods.(iv) If peptide assignments passed this scoring filter, the corresponding MS͞MS spectra were manually reviewed. For an assignment to be accepted the MS͞MS spectrum must have a continuous b-ory-ion series of at least five residues and the three most intense fragment peaks assigned to either an a-, b-, or y-ion, to an a-, b-, or y-ion resulting from a neutral loss of water or ammonia, or to a multiply protonated fragment ion. All review of peptide assignments and manual interpretation of MS͞MS spectra were facilitated by SCAFFOLD, a proteome software package.

PAPANO, a nitric oxide donor with defined release kinetics, or nonspecific labeling, ascorbate was omitted to largely prevent CysNO, an effective transnitrosating agent. Exposure of HASMC reduction of S-nitrosocysteine. Although ascorbate-independent to 100 ␮M CysNO for 20 min generated 3.0 Ϯ 0.3 nmol of protein biotin-HPDP labeling of S-nitrosocysteine is possible, naı¨ve and S-nitrosocysteine per mg of protein, whereas exposure to 2 mM cysteine-treated HASMC did not contain significant levels of PAPANO for 1 h generated 0.40 Ϯ 0.03 nmol of protein S- endogenous S-nitrosoproteins quantified by reductive chemistries

nitrosocysteine per mg of protein (mean Ϯ SD, n ϭ 4). These two coupled to chemiluminescence detection. Therefore, omission of MEDICAL SCIENCES conditions were used to explore the S-nitrosoproteome of ascorbate from these conditions served as an appropriate false HASMC. The difference in the yield of protein S-nitrosocysteine positive control. MS͞MS sequence-to-spectrum assignments from between CysNO and the nitric oxide donor treatment may reflect these treatments were evaluated by the same criteria as described the higher efficiency of S-nitrosylation by CysNO consistent with in Fig. 1 and were used to eliminate peptide identifications if they previous results (23). Cell culture studies have shown that exoge- were also identified in the NO-treated samples. A total of 18 nous CysNO is effectively transported intracellularly via the amino peptides belonging to 16 proteins were identified as possible false acid transporter system transporter system (23). Consequently, positives (Table 2, which is published as supporting information on intracellular CysNO may facilitate the formation of protein S- the PNAS web site), and therefore, were not considered targets of nitrosocysteine adducts primarily by replenishment of endogenous S-nitrosylation under our experimental conditions. S-nitrosoglutathione or by direct transnitrosation. In contrast, nitric oxide could be consumed by other cellular targets such as soluble Identification of Proteins and Sites of S-Nitrosylation by LC-MS͞MS. guanylate cyclase and thus a smaller fraction may participate in Employing selective peptide capture followed by LC-MS͞MS anal- S-nitrosative chemistries. Therefore, the treatment of HASMC ysis, 18 S-nitrosocysteine-containing peptides belonging to 16 pro- with either CysNO or PAPANO, followed by site-specific proteins were identified in HASMC exposed to CysNO (Table 1). The teomic analysis of protein S-nitrosocysteine formation, allowed us identification of S-nitrosylated proteins with diverse molecular to evaluate the potential selectivity of S-nitrosylation. weights and cellular roles such as cytoskeletal proteins, chaperones, proteins of the translational machinery, calcium-binding proteins, Evaluation of MS͞MS Sequence-to-Spectrum Assignments. Because and an ion channel protein supported the robustness of this of the selectivity of S-nitrosocysteine modification and the peptide technique. From the 16 proteins identified as potential targets of enrichment strategy used, rigorous selection criteria, as described in S-nitrosylation, 14-3-3 protein ␪, 14-3-3 protein ␨, annexin A2, Fig. 1, were used to identify peptides from the tandem MS elongation factor 2, and elongation factor 1 A-1 had been identified (MS͞MS) analysis. Typical MS͞MS spectra that either met or failed by the biotin switch method in various other systems (17–19, 24). In these criteria are depicted in Fig. 1. Importantly, the mass shift addition, Cys-137 of RAB3B has been proposed as susceptible to due to the Cys–N-[6(biotinamido)hexyl]-3Ј-(2Ј-pyridyldithio) pro- S-nitrosylation based on a conserved NKCD motif (25). Because pionamide (HPDP–biotin) adduct (ϩ428) was present in either the these experiments identified S-nitrosocysteine at residue 184, fur- y-orb-ion series for accepted peptide assignments. Although these ther work will be necessary to examine the site-specificity of selection criteria would minimize false peptide identifications re- S-nitrosylation in RAB3B. Additionally, four S-nitrosocysteine- sulting from MS͞MS sequence-to-spectrum assignments, they containing peptides belonging to four proteins were identified after would not prevent false positive peptide assignments arising from exposure to a nitric oxide donor (Table 1). Two of the proteins, biotin–HPDP labeling of cysteine residues that were not completely 14-3-3 ␨ and GRP75, were also identified as S-nitrosylated at the blocked by methyl methanethiosulfonate. As a control for this same residue after CysNO treatment, whereas microtubule-

Greco et al. PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 ͉ 7421 Downloaded by guest on October 1, 2021 Table 1. HASMC S-nitrosoproteome Uniprot Biological function, protein name accession no. Sequence† Residue‡ Z§ XCorr¶

Cell growth and maintenance Myosin heavy chain 9 P35579 KQELEEIC*HDLEAR 916 3 4.3 LQLQEQLQAETELC*AEAEELR 895 3 5.8 VEDMAELTC*LNEASVLHNLK 90 3 3.7 Vinculin Q5SWX2 VENAC*TK 85 2 2.8 Microtubule-associated protein 4࿣ P27816 C*SLPAEEDSVLEK 635 3 3.9 Signal transduction 14-3-3 protein ␨ P63104 YDDMAAC*MK 25 2 3.6 14-3-3 protein ␨࿣ P63104 YDDMAAC*MK 25 2 3.7 14-3-3 protein ␪ P27348 YDDMATC*MK 25 2 3.5 Annexin A2 Q567R4 GLGTDEDSLIEIIC*SR 133 3 4.2 Annexin A11 P50995 GVGTDEAC*LIEILASR 294 3 3.5 VAV-like protein Q6TPQ2 C*RSLSQGMELSC#PGSR 33 3 3.0 Protein metabolism Elongation factor 2 P13639 DLEEDHAC*IPIK 567 3 2.9 Elongation factor 1 A-1 P68104 DGNASGTTLLEALDC*ILPPTR 234 3 4.1 Eukaryotic initiation factor 5AII Q9GZV4 YEDIC*PSTHNMDVPNIK 73 3 4.0 T-complex protein 1, ␨ subunit P40227 NAIDDGC*VVPGAGAVEVAMAEALIK 405 3 5.0 Cyclophilin B P23284 DVIIADC*GK 194 2 3.1 GRP75 P38646 VC*QGER 487 2 2.4 GRP75࿣ P38646 VC*QGER 487 2 2.3 Transport COP-A Q8IXZ9 AWEVDTC*R 245 2 2.5 Ras-associated protein 3B P20337 LVDAIC*DK 184 2 3.1 Chloride intracellular channel 4 Q9Y696 DEFTNTC*PSDK 234 2 3.5 Nucleic acid metabolism Myoneurin࿣ Q8WX93 VSSC*EQR 740 2 2.7

Biotin–HPDP-labeled cysteine is indicated by an asterisk, methyl disulfide is indicated by a #. †CysNO-containing tryptic peptide sequences. ‡Residue numbers refer to UniRef database sequences (www.uniprot.org). §The charge state at the precursor peptide associated with the highest XCorr value. ¶The highest XCorr value obtained for that peptide assignment across four independent experiments. ࿣Proteins identified from PAPANO-treated HASMC. All other proteins were identified from CysNO-treated HASMC.

associated protein 4 and myoneurin were exclusive to PAPANO- Golgi complex (Fig. 2 B and C), consistent with the proposed treated HASMC. subcellular localizations of several of the identified proteins in The ability of this method to identify S-nitrosylated proteins from Table 1. Treatment with para-hydroxymercuricbenzoate, which as little as 0.4 nmol of S-nitrosocysteine per mg of protein is an displaces S-nitrosocysteine, significantly abolished S-nitrosocys- improvement in sensitivity and, hence, proteome coverage over the teine immunoreactivity (Fig. 2A). Of particular interest was the traditional biotin-switch approach. For example, exposure of RAW immunogold labeling located in close vicinity to the Golgi complex, 264.7 cells to 250 ␮⌴ CysNO generated Ϸ5.5 nmol of S- which was largely associated with membranes of the endoplasmic nitrosocysteine per mg of protein from which the standard biotin- reticulum and on vesicular membrane profiles near the Golgi (Fig. switch assay identified three S-nitrosylated proteins (24). This 2 B and C). Based on the proteomic data and the specific location increase in sensitivity was likely due to the enrichment of S- of these vesicles at lateral rims and cis-Golgi facing ER exit sites, nitrosocysteine-containing peptides and subsequent MS͞MS anal- these membranes could represent COP-I-coated vesicles. Immu- ysis using electrospray ionization and linear ion trap detection. nogold double labeling against S-nitrosocysteine and COP-I was Critically, the increase in sensitivity did not sacrifice selectivity, as performed, revealing low but distinct labeling on ER membranes nearly 90% (43 of 49) of the unique peptides that passed the (Fig. 2D) as well as occasional localization on vesicular membranes selection criteria contained a Cys–HPDP–biotin adduct. The cap- of the Golgi complex. (Fig. 2D, arrow). Recent studies have ture of six nonspecific peptides lacking a biotinylated adduct was suggested that, besides the COP-I vesicle coat, proteins of the 14-3-3 likely due to the harsher elution conditions required to denature family also recognize arginine-based ER localization signals on avidin and release the biotinylated peptides. Overall, the selectivity multimeric membrane proteins (26). Because this proteomic study and increased sensitivity of this method, as well as the ability to identified COP-A, 14-3-3 ␨, RAB3B, cyclophilin B, and chloride identify both the modified proteins and the sites of S-nitrosylation intracellular channel protein, which have proposed roles in ER͞ in a single experiment represent a significant advantage for eluci- Golgi transport and ER protein folding, this finding suggested a dating the S-nitrosoproteome in complex biological mixtures. regulatory role for S-nitrosylation in these cellular processes. In- terestingly, recent studies have revealed a role for S-nitrosylation in Evaluation of S-Nitrosylation in HASMC by Immunogold Electron the regulation of vesicular trafficking in endothelial and epithelial Microscopy. The cellular distribution of protein S-nitrosocysteine cells (4, 7), platelets (5), and neurons (6). In addition, nitric oxide was explored by high-resolution electron microscopy and immuno- has been identified as a proximal mediator of ER stress responses, gold labeling using monoclonal and polyclonal anti-S-nitrosocys- although the role of S-nitrosylation was not evaluated (27). teine antibodies. After treatment of HASMC with 100 ␮M CysNO, significant immunoreactivity for protein S-nitrosocysteine was ob- Sequence Analysis of S-Nitrosocysteine-Containing Peptides. The served in distinct cellular compartments such as the endoplasmic site-specific mapping of S-nitrosocysteine residues allowed direct reticulum membrane and vesicular membrane structures near the comparison of primary peptide sequences for motifs that may

7422 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600729103 Greco et al. Downloaded by guest on October 1, 2021 thought to contain S-nitrosocysteine, and therefore, they served as an appropriate peptide population for comparison. Sequence alignment of these 18 sequences revealed that at positions Ϫ3 and Ϫ4 acidic residues occurred at lower frequency, 34% and 17%, compared to 50% and 40% for the S-nitrosocysteine-containing peptides, respectively. Similarly, the frequency of basic residues at position 2 dropped to 6% compared to the S-nitrosocysteine- containing peptides (30%). Given the strong trend for flanking acidic͞basic residues revealed by alignment of S-nitrosocysteine- containing peptides, this finding provides some of the best direct evidence supporting the acid͞base motif. Another factor that may govern S-nitrosylation specificity is the occurrence of local hydrophobicity surrounding the cysteine residue (28). Construction of Kyte–Doolittle hydropathy plots revealed that the S-nitrosocysteine residues identified in T-complex protein 1, ␨ subunit, annexin A11, and elongation factor 1 A-1 were located in discrete motifs of increased hydrophobicity (Fig. 3C). Although primary sequence analyses are useful for determining structural features that underlie the specificity of posttranslational modifications, they do not reveal motifs that result from three- dimensional protein structure. Therefore, proteins identified in Table 1 and for which the crystal structures (Ͼ85% homology to the identified proteins) have been determined were evaluated for acid͞base motifs. Four of the 20 proteins, 14-3-3 ␨, 14-3-3 ␪, RAB3B, and chloride intracellular channel 4 met these criteria. Evaluation of the molecular models revealed that for each protein, an acid͞base motif opposing the identified cysteine was present within a molecular radius ranging from 2.4 to 7.1 Å (Fig. 4, which is published as supporting information on the PNAS web site). Because the proteomic studies identified 14-3-3 ␨ and GRP75 as targets of S-nitrosylation in both CysNO and PAPANO-treated HASMC, these agents may share similar molecular specificities with respect to protein S-nitrosocysteine formation. On the other hand, myoneurin and microtubule-associated protein 4, which were identified only from PAPANO-treated HASMC, did not contain acid͞base or hydrophobic motifs by primary sequence analysis. MEDICAL SCIENCES Also, the crystal structures for these proteins have not been determined. Therefore, the presence of common motifs for some Fig. 2. High-resolution immunoelectron microscopy. HASMC exposed to 100 but not all proteins identified from CysNO and PAPANO treat- ␮M CysNO for 20 min were ﬁxed and processed for EM. Immunoreactivity for ments suggests that protein S-nitrosocysteine formation derived S-nitrosocysteine-containing proteins was visualized by 10-nm protein A gold from the nitric oxide radical donor include both secondary reac- particles. COP-1 immunoreactivity was visualized by 15-nm protein A gold tions of nitric oxide to generate transnitrosating species as well as particles. (A) Sections were treated with para-hydroxymercuricbenzoate to other potential chemistries (8, 9). displace the S-nitrosocysteine adducts and then stained with monoclonal In summary, the proteomic approach used permitted not only the anti-S-nitrosocysteine antibody (26). (B) S-nitrosocysteine immunoreactivity evaluation of the S-nitrosoproteome in HASMC, but facilitated the (monoclonal antibody) was associated with endoplasmic reticulum (er) and elucidation of two S-nitrosylation motifs that govern the selectivity small vesicular structures (arrows) in the vicinity of the Golgi complex (g). (C) A similar pattern of staining obtained with a polyclonal anti-S-nitrosocysteine of modification. By systematically evaluating potential peptide antibody (asterisk indicates labeling of small vesicle). (D) Double labeling for sequence-to-spectrum assignments and by eliminating false positive S-nitrosocysteine (10-nm gold) and COP-1 (15-nm gold) showed localization S-nitrosocysteine-containing peptide identifications, 20 unique S- on vesicular membrane proﬁles (arrow). (Scale bar, 200 nm.) nitrosocysteine-containing peptides belonging to 18 proteins were identified. The identification of cytoskeletal, signal transduction, and ER-associated proteins implicates S-nitrosylation in the regu- govern S-nitrosylation specificity. It has been proposed that there is lation of smooth muscle cell proliferation, apoptosis, and ER a predisposition toward flanking basic (Lys, Arg, His) and acidic protein folding. The detection of proteins that participate in the (Asp, Glu) residues, and if positioned within6Åofthemodified ER͞Golgi transport system is consistent with previous reports cysteine, these residues could regulate S-nitrosylation and denitro- implicating S-nitrosylation in the regulation of vesicular trafficking sation by altering thiol nucleophilicity (1). Sequence alignment of in other cell types (4–7). Significantly, through regulation of vas- the 18 S-nitrosylated peptides identified from CysNO-treated cular smooth muscle ER͞Golgi function, S-nitrosylation may in- smooth muscle cells revealed that the highest occurrence of acidic fluence vascular wall stress responses. (D, E) residues was Ϸ50% and 40%, at positions Ϫ3 and Ϫ4, respectively, relative to the modified cysteine. The highest occur- Materials and Methods rence of basic (K, R, H) residues was Ϸ30% at position 2 (Fig. 3A). Chemicals and Reagents. Unless otherwise indicated, chemicals were Interestingly, there were no basic residues in position Ϫ3 and Ϫ4, purchased from Sigma. Kaighn’s modification of Ham’s F12 me- and acidic residues at position 2 only occurred at a 10% frequency. dium with 2 mM L-glutamine (F12K), Earle’s Balanced Salt Solu- Given the relatively small number of peptides being compared, the tion (EBSS), and SDS͞PAGE 4–12% Bis-Tris gradient gels were differences observed may result by chance; therefore, the same purchased from Invitrogen. Micro Bio-spin P6 columns were analysis was performed for the 18 false-positive peptide identifica- obtained from Bio-Rad. PAPANO was purchased from Cayman tions (Fig. 3B). These peptides were excluded because they were not Chemicals. Biotin-HPDP and streptavidin–agarose were purchased

Greco et al. PNAS ͉ May 9, 2006 ͉ vol. 103 ͉ no. 19 ͉ 7423 Downloaded by guest on October 1, 2021 Fig. 3. S-nitrosylation specificity motifs. (A) Sequence alignments of 18 S-nitrosocysteine-containing peptides identified from CysNO-treated HASMC comparing the occurrence of amino acids at positions flanking the modified cysteine. (B) Sequence alignments of 18 false positive peptides comparing the occurrence of amino acids at positions flanking the cysteine residue. (C) Kyte–Doolittle hydropathy plots from regions flanking the identified S-nitrosocysteine residue (arrow). The identified S-nitrosocysteine residues from T-complex protein 1, ␨ subunit (Left), annexin A11 (Center), and elongation factor 1 A-1 (Right) were located within hydrophobic pockets. Hydropathy plots were constructed by using a window of 13 aa.

from Pierce. Ultrafree-MC filters, PVDF Immobilon-FL, and Quantitation of Protein S-Nitrosocysteine. Intracellular S-nitroso- ZipTipC18 P10 were from Millipore. Trypsin Gold (mass spectrom- protein content was determined from HASMC cellular lysates by etry grade) was purchased from Promega. Mouse monoclonal and chemiluminescence using a Sievers 280 nitric oxide analyzer. rat polyclonal anti-nitrosocysteine antibodies were obtained from HASMC were treated with NO agents as described above, and cell A.G. Scientific. extracts were obtained as described below. Lysates were then passed CysNO was prepared by mixing equimolar amounts of L-cysteine over two successive Micro Biospin P6 columns to remove low and NaNO2 under acidic conditions (0.25 M HCl) in the presence molecular weight S-nitrosothiols, and protein concentration was of 0.1 mM DTPA. CysNO stock solutions (500 mM) were prepared determined. Lysates were incubated with 0.1% SNA͞10% glacial fresh. The final concentration of CysNO was determined from acetic acid for at least 15 min to remove nitrite contamination. absorbance at 334 nm by using the extinction coefficient 900 Ϸ0.12 mg of cellular lysates were routinely injected into the reaction MϪ1⅐cmϪ1. Immediately before exposure to cell cultures, an inter- vessel containing 5 ml of 60 mM potassium iodide (KI) and 10 mM mediate dilution of the stock solution was prepared in HEN buffer iodine (I2) in glacial acetic acid at 37°C. Under these conditions, the (250 mM Hepes, pH 7.7͞1 mM EDTA͞0.1 mM neocuproine). lower limit of detection was 0.03 pmol of SNO per mg of total PAPANO was prepared as a concentrated stock in 0.01 M NaOH, protein. Equivalent results for S-nitrosoprotein content were also and the final concentration was determined by absorbance at 250 obtained by using Cu(I)͞Ascorbate reduction method. As a nega- nm using the extinction coefficient 8050 MϪ1⅐cmϪ1. All stock tive control for detection of S-nitrosocysteine, lysates were incu- solutions were stored on ice in the dark. bated with 3.5 mM HgCl2 for 20 min at 4°C.

Cell Culture and Treatment with NO Agents. HASMCs were obtained Cell Extract Preparation and Biotin Switch Assay. Unless otherwise at passage 15 or 16 from American Type Culture Collection indicated, all steps were performed in the dark. After the treatment (Manassas, VA) and cultured in F12K supplemented with 10 mM medium was removed, the cells were quickly trypsinized at 37°C, Hepes, 10 mM TES, 10% FBS, ITS (0.01 mg/ml insulin͞0.01 mg/ml inactivated with F12K containing 0.1% FBS, and centrifuged at transferrin͞10 ng/ml sodium selenite), 0.03 mg͞ml ECGS, and 0.05 130 ϫ g for 6 min at 4°C. Cell pellets were washed three times with mg͞ml ascorbic acid. Cells were maintained in a 5% CO2 incubator ice-cold PBS containing 1 mM EDTA and 0.1 mM neocuproine. at 37°C in T175 flasks. Experiments were performed between The biotin-switch assay was performed with between 0.5 and 1 mg passages 16 and 21. When intact cells were ready for analysis of cellular protein as described (15) with minor modification. Cell (Ϸ85% confluency) they were washed twice with EBSS and pellets were resuspended in lysis buffer (HEN buffer containing 1% incubated in the dark for 20 min with 100 ␮M L-cysteine or 100 ␮M Triton X-100). CysNO at 37°C. For PAPANO treatments, cells were exposed to 2 Resuspended pellets were then centrifuged at 12,000 ϫ g, 4°C for mM PAPANO for1hat37°C in FBS͞ITS͞Asc-free medium (basal 10 min. The biotin switch assay was performed with between 0.5 media) or in basal media alone as a control. and 1 mg of protein. The lysates were adjusted to 0.5 mg͞ml

7424 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600729103 Greco et al. Downloaded by guest on October 1, 2021 containing 2.5% SDS and 200 mM methyl methanethiosulfonate flow set at 700 nl͞min. The mobile phase consisted of 0.1% formic and incubated at 50°C for 20 min, vortexing every 4 min to block acid in either HPLC grade water (A) or acetonitrile (B). Peptides free thiols. After blocking, cell extracts were precipitated with two were eluted initially with 99% A, then 95% A for 3–5 min, then a volumes of Ϫ20°C acetone, incubated at Ϫ20°C for 20 min, cen- linear gradient to 72% A by 33 min, then to 20% A at 40 min and trifuged at 12,000 ϫ g, 4°C for 10 min, washed four times with held to 45 min, then to 99% A at 52 min and held until 60 min. acetone, and resuspended in 0.2 ml of HENS buffer (25 mM Hepes, MS͞MS spectra were acquired by using a full scan, which was pH 7.7͞0.1 mM EDTA͞0.01 mM neocuproine͞1% SDS). To the followed by four data-dependent scans on the four most intense blocked proteins, 0.4 mM biotin-HPDP and 5 mM ascorbate were precursor ions. Precursors that were detected twice within 15 s were added and incubated at 25°C for 1 h while rotating. To control for put on a dynamic exclusion list for a period of 60 s. MS͞MS spectra nonspecific HPDP labeling of unmodified cysteines, ascorbate was were matched to human NCBI RefSeq database sequences with omitted. After incubation, proteins were precipitated with acetone SEQUEST (Bioworks Browser 3.1 SR1) (Thermo Electron, San Jose, as described above. Samples in which protein digestion was per- CA). Cysteine modification by methyl methanethiosulfonate (ϩ46 formed were resuspended in 0.45 ml of 0.1 M ammonium bicar- atomic mass units) and by biotin-HPDP (ϩ428 atomic mass units) bonate and 0.5% SDS. Protein concentration was checked by the was specified as variable modifications. For further details, see BCA assay (Pierce). Supporting Text and Figs. 5–23, which are published as supporting information on the PNAS web site. Protein Digestion and Affinity Peptide Capture. Biotinylated proteins were incubated with trypsin (1:30 enzyme͞protein ratio) at 37°C for Immunoelectron Microscopy. HASMC cells were treated with 18–24 h in the dark. Samples were then passed through Ultra- CysNO as described above and immediately fixed in 2% para- free-MC 10-kDa cutoff filters that had been previously rinsed with formaldehyde (PFA) and 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for2hatroom temperature. Fixed methanol and washed with H2O. The filtrate containing the peptides was recovered and incubated with Ϸ50 ␮l of dry, washed cells were stored at 4°C in 1% PFA until cyrosectioning. Ϫ streptavidin–agarose beads per mg of initial protein for 30 min with Fifty-nanometer-thick cryosections were cut at 120°C by using gentle mixing. Samples were centrifuged at 5,000 ϫ g for 5 min, and an Ultracut S ultramicrotome (Leica). The sections were col- the supernatants were discarded. The beads were washed five times lected on carbon-coated formvar grids using a mixture of 1.8% with 10 volumes of 1 M ammonium bicarbonate, followed by five methylcellulose and 2.3 M sucrose (29) and incubated with washes with 10 volumes of deionized water. Between washes, primary nitrosocysteine antibodies (30) and 10 nm of protein A samples were centrifuged at 1,000 ϫ g for 1 min. Elution buffer gold (31). After labeling, the sections were fixed with 1% containing 70% formic acid (FA) was incubated with the beads for glutaraldehyde, counterstained with uranyl acetate, and embed- 30 min with gentle mixing. The captured peptides were recovered ded in methylcellulose–uranyl acetate. The specificity of the by centrifuging beads at 5,000 ϫ g for 4 min and collecting the labeling was verified in control experiments where sections were supernatant. To ensure complete removal of streptavidin–agarose, treated with 3.5 mM p-hydroxymercuricbenzoate for 30 min the samples were centrifuged again. The captured peptides were (three 10-min treatments). Immunogold double labeling was evaporated to Ϸ5 ␮l in vacuo, resuspended in 20 ␮l of 0.1% FA, and performed by using 10 and 15 nm of protein A gold. After labeling, the sections were fixed with 1% glutaraldehyde, coun- desalted by using Zip-Tips. terstained with uranyl acetate, and embedded in methyl cellu- lose–uranyl acetate. The sections were viewed in a JEOL MEDICAL SCIENCES Analysis by LC-MS͞MS. Desalted samples were analyzed on a 1200CX electron microscope. Thermo LTQ linear trap instrument equipped with a Thermo micro electrospray source, and a Thermo Surveyor pump and We are grateful to Sheryl Stamer for her assistance with mass spec- autosampler (Thermo Electron Corporation, San Jose, CA). LC- ͞ ͞ trometry and MS MS data analysis. We thank the Protein Core at the MS MS analyses were done by reverse phase chromatography on Stokes Research Institute for their assistance with SEQUEST analysis and an 11-cm fused silica capillary column (100 ␮m i.d.) packed with the Proteome Software development team for their technical assistance Monitor C-18 (5 ␮m) (Column Engineering, Ontario, CA) with the and expertise with SCAFFOLD.

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